CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug-resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus-1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets. Since its discovery as an adaptive immune system in prokaryotes, the CRISPR/Cas9 system has been developed into a multi-faceted genetic modification tool, which can now be used to induce gene deletions or specific gene insertions, such as conditional alleles or endogenous reporters in virtually any organism. The generation of CRISPR/Cas9 libraries that can be used to perform phenotypic whole genome screens provides an important new tool that will aid in the identification of critical host factors involved in the pathogenesis of infectious diseases. In this review, we will discuss the development and recent applications of the CRISPR/Cas9 system used to identify novel regulators, which might become important in the fight against infectious diseases.

Insensitivity of perturbed carboxyl pK(a) values in the ovomucoid third domain to charge replacement at a neighboring residue.

A number of carboxyl groups in turkey ovomucoid third domain (OMTKY3) have low pK(a) values. A previous study suggested that neighboring amino groups were primarily responsible for the low carboxyl pK(a) values. However, the expected elevation in pK(a) values for these amino groups was not observed. In the present study, site-directed mutagenesis is used to investigate the origins of perturbed carboxyl pK(a) values in OMTKY3. Electrostatic calculations suggest that Lys 34 has large effects, 0.4-0.6 unit, on Asp 7, Glu 10, and Glu 19 which are 5-11 A away from Lys 34. Two-dimensional (1)H NMR techniques were used to determine pK(a) values of the acidic residues in OMTKY3 mutants in which Lys 34 has been replaced with threonine and glutamine. Surprisingly, the pK(a) values in the mutants are very close to those of the wild-type protein. The insensitivity of the acidic residues to replacement of Lys 34 suggests that long-range electrostatic interactions play less of a role in perturbing carboxyl pK(a) values than originally thought. We hypothesize that hydrogen bonds play a key role in perturbing some of the carboxyl ionization equilibria in OMTKY3.

Bactericidal activity of mammalian cathelicidin-derived peptides.

Endogenous antimicrobial peptides of the cathelicidin family contribute to innate immunity. The emergence of widespread antibiotic resistance in many commonly encountered bacteria requires the search for new bactericidal agents with therapeutic potential. Solid-phase synthesis was employed to prepare linear antimicrobial peptides found in cathelicidins of five mammals: human (FALL39/LL37), rabbit (CAP18), mouse (mCRAMP), rat (rCRAMP), and sheep (SMAP29 and SMAP34). These peptides were tested at ionic strengths of 25 and 175 mM against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and methicillin-resistant Staphylococcus aureus. Each peptide manifested activity against P. aeruginosa irrespective of the NaCl concentration. CAP18 and SMAP29 were the most effective peptides of the group against all test organisms under both low- and high-salt conditions. Select peptides of 15 to 21 residues, modeled on CAP18 (37 residues), retained activity against the gram-negative bacteria and methicillin-sensitive S. aureus, although the bactericidal activity was reduced compared to that of the parent peptide. In accordance with the behavior of the parent molecule, the truncated peptides adopted an alpha-helical structure in the presence of trifluoroethanol or lipopolysaccharide. The relationship between the bactericidal activity and several physiochemical properties of the cathelicidins was examined. The activities of the full-length peptides correlated positively with a predicted gradient of hydrophobicity along the peptide backbone and with net positive charge; they correlated inversely with relative abundance of anionic residues. The salt-resistant, antimicrobial properties of CAP18 and SMAP29 suggest that these peptides or congeneric structures have potential for the treatment of bacterial infections in normal and immunocompromised persons and individuals with cystic fibrosis.

Theoretical and experimental analysis of ionization equilibria in ovomucoid third domain.

2D-NMR experiments were used to determine the pKa values ranging from 8.0 to >/=11.1 of seven basic residues in turkey ovomucoid third domain (OMTKY3) and were compared to values predicted as described by Antosiewicz et al. [(1996) Biochemistry 35, 7819-7833]. Lys 13, 29, and 34 were previously attributed with increasing the acidity of numerous acidic residues [Schaller, W., and Robertson, A. D. (1995) Biochemistry 34, 4714-4723]. These interactions were expected to raise the pKa values of those basic groups; however, the pKa values of Lys 13 and 34 are less than the model compound values. The pKa values of the other basic residues are greater than the model compound values and, unlike the acidic residues, all are surprisingly insensitive to salt. While the calculations properly predict the direction of most of the pKa shifts and provide valuable insight into the possible molecular origins of the interactions that perturb pKa values, there is a tendency to overestimate the magnitude of the shifts and their salt dependence. Interestingly, the shapes of both the calculated and observed transitions are often more complex than expected for a simple titration, suggesting that pKa values at many sites are changing during the transition. Differences between predicted and experimental pKa values and titration profiles for some residues may be due to as yet uncharacterized structural changes at the extremes of pH.

Isolation of very virulent Marek's disease viruses from vaccinated chickens in Australia.

Very virulent Marek's disease viruses (vvMDV), defined as isolates against which the herpesvirus of turkey (HVT) vaccine provide poor protection, have been isolated from poultry flocks in both the United States and Europe. Twenty-one samples from vaccinated Australian flocks, experiencing problems with excessive Marek's disease (MD), were tested for the presence of transmissible MD viruses (MDV). Of the 16 samples which contained a transmissible agent, 14 were pathogenic in chickens, based on the development of MD lesions or depression of the bursa/body weight ratio. Of the pathogenic isolates which have been successfully typed 10 were serotype 1, and one was serotype 2 MDV. Pathogenicity of isolates varied. Several isolates caused tumours in 20-30% of both vaccinated and unvaccinated chickens. Two isolates, MPF6 and MPF23, caused tumours in more than 50% of chickens. When MPF6 and MPF23 were tested in vaccine trials bivalent vaccine gave no better protection against development of MD lesions than a monovalent vaccine. Isolate MPF23 was so pathogenic that lesions were produced in all chickens, regardless of the vaccine protocol used. Therefore vvMDV have been isolated in Australia, and unlike the vaccines tested overseas, bivalent Australian vaccines do not appear to provide greater protection against these vvMDV.

A survey of the pectic content of nonlignified monocot cell walls.

The primary cell walls of graminaceous monocots were known to have a low content of pectin compared to those of dicots, but it was uncertain how widespread this feature was within the monocots as a whole. Nonlignified cell walls were therefore prepared from 33 monocot species for determination of their pectin content. It was not possible to solubilize intact pectins quantitatively from the cell walls, and the pectin content was assessed from three criteria: the total uronic acid content; the content of alpha-(1,4')-D-galacturonan isolated by partial hydrolysis and characterized by electrophoresis and degradation by purified polygalacturonase; and the proportion of neutral residues in a representative pectic fraction solubilized by sequential beta-elimination and N,N,N'N'-cyclohexanediaminetetraacetic acid extraction. Low galacturonan contents were restricted to species from the Gramineae, Cyperaceae, Juncaceae, and Restionaceae. Other species related to these had intermediate galacturonan contents, and the remainder of the monocots examined had high galacturonan contents comparable with those of dicots. The other criteria of pectin content showed the same pattern.

Bovine leptospirosis: microbiological and histological findings in cattle at slaughter.

Kidneys from cattle at slaughter were examined for the presence of leptospires. Of 218 (8.3%) kidneys leptospires were isolated from 18; all were identified as Leptospira interrogans serovar hardjo. None of the leptospire-infected kidneys had histopathological lesions indicative of leptospirosis and leptospires were demonstrated in only 2 by immunogold silver staining. Leptospires infected kidneys remained viable for at least 21 days when stored at 4 degrees but became non-viable within 14 days when stored frozen at -15 degrees.

Systemic mycotic disease of captive crocodile hatchling (Crocodylus porosus) caused by Paecilomyces lilacinus.

The sudden death occurred of a captive Estuarine crocodile hatchling (Crocodylus porosus). On autopsy, granuloma-like lesions were seen in the liver, left lung and spleen, and branching, septate fungal hyphae were observed in sections of liver and spleen. The fungus isolated from the liver showed characteristics of both Paecilomyces lilacinus and Paecilomyces marquandii but was closer to the former species. This is apparently the first report of the isolation of this fungus from a reptile in Australia.

Experimental reproduction of the runting-stunting syndrome of broiler chickens.

A model for the reproduction of the runting-stunting syndrome (RSS) of broiler chickens is described. In this model, groups of at least 90 day-old broiler chickens were inoculated (per os) with various tissue homogenates or virus preparations. During the first week post-inoculation, birds were examined for the development of histopathological changes in their intestines. At day 14 post-inoculation, the remaining birds were weighed and tested for elevations in plasma amylase activity and examined for the development of pancreatic atrophy. Bacteria-free intestinal and pancreatic homogenates from chickens of different ages, taken from flocks which developed RSS, regularly induced a lower mean live-weight in treated birds. Of these, only intestinal homogenates prepared from 5-day-old birds induced intestinal lesions, lowered mean live-weight and increased the incidence of both elevated plasma amylase activity and pancreatic atrophy. These changes were more marked in birds exposed to short periods of sub-optimal temperatures during the first week post-inoculation. An ultracentrifuged pellet prepared from this intestinal homogenate, was also found to induce an increased incidence of pancreatic atrophy in treated birds. These studies suggest that the causative agent(s) of RSS is an as yet unidentified virus, and that the effects of this infection are greater in birds subjected to stress, such as sub-optimal temperature exposure, within the first week of hatch.

Toxicity episodes involving agricultural chemicals and other substances in birds in Victoria, Australia.

A series of case reports detailing observations on toxicity episodes in birds caused by a variety of agricultural chemicals and other substances is presented. These problems arose as a result of ignorance, accident and malicious intent. The episodes involved maldison, monocrotophos, fenitrothion, trichlorofon, dieldrin, chlordane, endrin, metaldehyde, bromadiolone, arsenic, lead and zinc. An unresolved episode where toxicity was implicated is also included.

Absence of a role for selenium deficiency in the runting syndrome of broiler chickens in Australia.

The role of selenium deficiency in the etiology of the runting-stunting syndrome (RSS) of broiler chickens in Australia was investigated. Commercial broiler chickens maintained on selenium-deficient developed signs consistent with selenium deficiency of exudative diathesis and markedly reduced plasma glutathione peroxidase activity, but they did not develop pancreatic atrophy and fibrosis or elevated plasma amylase activity, which are the other lesions associated with RSS. Supplementation of the diets of birds from a RSS-susceptible flock with a mixture of selenium, vitamin E, cysteine, and sulfate had no effect on the incidence of runting in the treated birds. In field outbreaks of RSS there were no observable differences between affected and unaffected birds in the concentration of selenium in tissue samples. Furthermore, evidence is presented which suggests that in cases of RSS, pancreatic atrophy and elevations in plasma amylase precede reductions in plasma glutathione peroxidase activity.

Investigations of toxicity episodes involving chemotherapeutic agents in Victorian poultry and pigeons.

This series of case reports details observations on toxicity episodes in poultry due to a variety of chemotherapeutic agents. These problems arose owing to overdosage, variation in species susceptibility, potentiation of the toxic effects of one substance by the presence of another substance, and particular disease or other on-farm factors. Ignorance and accident were responsible for some of these situations. The episodes involved monensin, salinomycin, nicarbazin, sulphaquinoxaline, dinitolmide, dimetridazole, nitrofurans, streptomycin, and 3-nitro-4-hydroxyphenylarsonic acid.

Field, clinical and pathological observations of a runting and stunting syndrome in broilers.

Observations on a runting and stunting syndrome in broiler chickens in Victoria, Australia, based on general observations from 1980 to 1983 on 2244 chickens from 109 affected broiler chicken flocks, are summarised. The details on 156 of these birds from five affected flocks with varying runting percentages are presented. Typically affected birds were presented with atrophy of the pancreas, the thymus and the bursa of Fabricius.

Detection of Clostridium difficile toxin with McCoy cell monolayers and cell suspensions and comparison with HeLa cell assay.

McCoy cell monolayers were compared with HeLa cell monolayers for the detection of Clostridium difficile toxin in 301 stool samples. Tests were positive (greater than or equal to 1/100 dilution) in 83 and 81 specimens tested with McCoy and HeLa cell monolayers, respectively. McCoy cell suspensions were compared with HeLa cell monolayers in 532 stool filtrates. Overall, 90 positive specimens were within one dilution and 432 filtrates were negative with either test, giving a correlation coefficient of r = 0.98. McCoy cell monolayers or suspensions may be a satisfactory substitute for the detection of C. difficile toxin in clinical specimens.

Blood glutathione peroxidase activity in horses in relation to muscular dystrophy and selenium nutrition.

The activity of glutathione peroxidase, a selenium containing enzyme, was measured in the blood of horses to determine its usefulness as an indicator of selenium status. In 15 horses the enzyme activity was positively related to the blood selenium concentration (P less than .001, r-0.98) over the range of enzyme activities of 8.2 to 140 units (mumoles NADP-oxidised/min/gHb) and selenium concentrations of 0.24 to 2.74 mumol/l. In a group of 8 horses which 2 foals had died with lesions of muscular dystrophy the enzyme activity increased from a mean of 11.8 units before treatment with selenium to 34.5 units after 2 intravenous injections of sodium selenite given one month apart. Another group of 8 horses grazing paddocks adjacent to this affected group did not receive any selenium treatment and had a mean enzyme activity of 11.9 units. Blood glutathione peroxidase activity was measured in 50 pasture-fed horses and 180 stall-fed horses. The range of activities found (7 to 158 units) indicated that selenium intake in horses varied widely between localities. All pasture-fed horses grazing areas where muscular dystrophy had occurred in foals had low activities (less than 20 units). In stall-fed horses the enzyme activity was influenced by selenium treatment, and horses which had been treated usually had higher activities than horses in the same stable with no history of selenium treatment. It was concluded that blood glutathione peroxidase is a suitable indicator of selenium status in horses.